An increased release of intracellular enzymes, indicative of cell death, has been observed upon reoxygenation of cardiac tissue following a sustained period of hypoxia (oxygen paradox). Two distinct pathways have been proposed to explain this phenomenon: a) the energy-dependent breakdown of cells injured during the preceding hypoxia, or b) the induction of additional damage by oxygen metabolites. Although evidence is available that oxygen radicals are formed upon reoxygenation and that antioxidants can protect against reperfusion injury, the presence of oxidative damage is not well established. The overall purpose of this project is to investigate some selected indices of oxidative stress in isolated-perfused heart tissue. Hearts from rats (which contain xanthine oxidase, a purported source of oxygen radicals) and from rabbits (which, like humans, lack this enzyme activity) will be used. Hearts will be perfused (Langendorff) for 30 min with oxygenated medium followed by 60 min of hypoxia and a further 30 min of oxygen. Cardiac oxygen uptake, lactate dehydrogenase release, left ventricular pressure and coronary flow rates will be monitored. The following indices of oxidative stress will be measured: a) the activity of sarcolemmal and sarcoplasmic reticulum (SR) calcium ATPase (this enzyme has been found to be susceptible to irreversible oxidative inhibition), b) alpha-tocopherol and alpha-tocopheryl quinone content, c) mixed disulfide and protein sulfhydryl content of whole heart, sarcolemmal and SR membranes, and d) cardiac conjugated diene and lipid hydroperoxide levels. Analyses will be performed in fresh heart, hearts perfused for 94 min with oxygenated medium, hearts perfused for 30 min with oxygenated medium followed by 60 min of hypoxia or 60 min of hypoxia plus 4 min of reoxygenation. Together, these studies will determine whether oxidative processes, which have been associated with cell death, are occurring in reoxygenated heart tissue. Preliminary data showed that cystamine, which oxidizes sulfhydryl groups and inhibits calcium ATPase in liver, can prevent myocyte lysis at reoxygenation. The effect of cystamine on sarcolemmal and SR protein sulfhydryls, mixed disulfides and calcium ATPase will be assessed. The role of calcium ATPase in controlling myocardial cell lysis at reoxygenation will be further studied by assessing the effect of 2 additional inhibitors of this enzyme, diamide and vanadium, on cardiac LDH release. Since inhibitors of ATP synthesis prevent enzyme release at reoxygenation, the effect of diamide, cystamine, and vanadate on myocardial energy content will also be measured. Finally, studies are proposed to investigate the ability of liposomally-entrapped ATP to mimic reoxygenation injury when infused 60 min of hypoxia and to prevent the oxygen paradox when infused throughout the hypoxic period. These studies will show whether the administration of exogenous ATP in a form which will enter cells is capable of protecting heart tissue from hypoxic injury or of stimulating enzyme release in hypoxic heart tissue not reoxygenated. Protection from hypoxic injury would have implications for improved organ preservation technology.